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Lecture 9

Lecture 9

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University of Toronto Scarborough
Biological Sciences
Dan Riggs

Lecture 9 - Nuclear and Chromatin Structure SPs: Figs 12-1, 2, 6, 7, 8, 12 Vocabulary: nuclear pore complex/nuclear matrix/chromatin/nuclear envelope/ nucleoporins/ nuclear localization signal/importins/ histones/ nucleosomes/ scaffold/ topoisomerases Talk about what will happen in the nucleus. Figure 12 – 1 This is a cartoon of the nucleus. The nucleus is a double membrane bound organelle but there is a lot of discontinuity within the membrane structure where membrane pores exist. At the membrane pore, transport is highly regulated. In the center of the nucleus there is a structure called the nucleolus where ribosomal genes are clustered, TRNAs and the beginnings of transcription occur. The cytoplasm or fluid-based part of the nucleus is called the nucleoplasm but there is also a structural framework of the nucleus referred to as the nuclear matrix. Chromatin is a general term which means a protein DNA complex so basically talking about chromosomes when you are talking about chromatin. When people think of chromosomes they think of long pieces of DNA which is true but there is an equal part of protein that makes up chromosomes so chromatin as a chromosome consists of equal parts of DNA and protein. Some examples of chromatin associated proteins are transcription factors that associate with DNA to help with transcription. Some structural proteins of chromatin are known as histones. Chromatin: DNA/protein complexthat constituteschromosomes ApproximatelyequalamountsofDNA/protein Proteins include 'structural' proteins such as histones and proteins involved in DNA metabolism (e.g. TFIID) Figure 12 – 2 A cross-section of a nuclear pore. Located inside the nucleus juxtaposed to the nuclear membrane is the nuclear lamina Which is a series of fibers which exist just under the nuclear surface. The lamina is composed of intermediate filaments called lamins. The nuclear envelope also has sites where chromatin is attached. You can see fibers that exist in the nucleus that are not arranged randomly but have specific territories that connect to things like the lamina. Figure 12 – 3/5 An electron micrograph of what the lamina looks like. It is sort of like an interwoven network of threads. The lamina serves as a structural barrier or structural support system for the nuclear envelope and also as an attachment site for chromatin fibers. It is remarkable because during meiosis or mitosis, As the chromatin condenses, the nuclear membrane must breakdown so this meshlike network must dissolve every cell cycle and then increase again once the nucleus reforms. Electron microscopy using freeze fracturing of the nuclear pores. The cytoplasmic face of the nuclear envelope is full of little, regular type structures with an octagonal shape made of eight subunits arranged in a circle. On the nuclear face of the nuclear envelope, you still see the same little structures but with a different shape (like a net). Which implies that there is a sideness to the structure. Figure 12 – 6 This image shows the cytoplasmic side at the top and the nucleoplasm side at the bottom. The nuclear pore complex is made up of a series of proteins collectively called nucleoporins. There are about 30 nucleoporins with about 8 copies of each. This gives the Nuclear pore complex an octagonal shape. In green is the membrane structure of the nucleus, there is also a cytoplasmic ring facing the cytoplasm, and a nuclear ring facing the nucleus. On the outside there are eight cytoplasmic filaments, and on the inside there are eight fibers which join together to form a nuclear basket. The important part of the nuclear pore complex is the actual pore itself. The central channel is open but the fibers in the middle of the pore are the proteins which are unstructured domains that move into the center and block the pore. These fibers can be rearranged to allow passage of things in and out through the channel. There is a serious amount of heavy traffic that moves in an out of the nuclear pore. Most active cells have around 10 million ribosomes. Ribosomes are composed of several ribosomal RNAs and numerous proteins some which reside in the nucleus and some which reside in the cytoplasm. This means that over half a million ribosomal proteins and around 14,000 ribosomal subunits are moved across the membrane every minute. RNA and RNA protein complexes move out into the cytoplasm. Proteins that are involved in maintaining the structure of the chromosomes, involved in DNA replication, and in transcription move in from the cytoplasm to the nucleus. So nuclear pores are very active portals for which proteins move through. How do proteins "know" to go to the nucleus or if they are in the nucleus how do they know to go to the cytoplasm? Heavy traffic! 7 If cell has 10 ribosomes, must import ~ 560,000 ribosomal proteins and export 14,000 ribosomal subunits every minute! RNAs and RNA/protein complexes move out Proteins move in (e.g. enzymes associated with DNA replication and transcription). How do proteins 'know' to go to the nucleus? There are address codes on them. Nuclear pore complexes are where small proteins can move freely across however the proteins must be less than 40 000 Daltons that is the molecular weight cut off for something to diffuse from one side to the other. Large protein like DNA polymerize have to have something to help them get to the other side. These larger proteins possess specific signals known as nuclear localization signals (NLS). Positively charged amino acids are important. They can either be in one region of the molecule or in several regions of the molecule and when the molecule folds it creates something that looks like a nuclear localization signal which is recognized by a specific chamber that says this is the protein that needs to go to the nucleus. Then, molecules that carry the nuclear localization signals, large proteins for example, interact with the receptors that assist their transport which looks something like the image on figure 12 – 7. Small proteins can freely diffuse into the nucleus ≈40,000 Daltons is the 'cut- off' Larger proteins possess specific signals Nuclear localization signal (NLS): one to several different regions of a protein that contain short stretches of positively charged amino acids Figure 12 – 7 The nucleoplasm is at the top and the cytoplasm is at the bottom. We want to get the red NLS-containing protein (the cargo) from the cytoplasm to the inside of the nucleoplasm. There are a class of proteins of which there are multiple genes and they are collectively referred to as importins. These are important for importing molecules into the nucleus. Importins are heterodimers - hetero means different and dimer means two. There is an alpha form seen in yellow and a beta form which is seen in green. What happens is that these dimers recognize the cargo molecules by virtue of the nuclear localization signal and they form a complex. By interacting and forming a complex, the dimers are able to interact with the cytoplasmic filaments that exist at the end of the pore complex (seen in panel 2). Then there is a conformational change that allows the cargo and the importins to move through the central channel and they find themselves in the nucleoplasm (panel 4). The cargo is now delivered however we need the importins to let go of the cargo so a protein inside the nucleus known as RAN which is a GTP binding protein interacts with importin beta. This induces a conformational change which makes the importin beta release the cargo and releases importin Alpha. RAN exports importin beta back to the cytoplasm where GTP is cleaved and makes RAN let go of importin beta. To get importin alpha back to the cytoplasm, another class of molecules known as exportins are needed. These recognize molecules in the nucleus that need to go back to the cytoplasm (which could be proteins or RNA complexes) and they move them to the cytoplasm by interacting with what are known as nuclear export signals. These are different from the nuclear localization signals which tend to be short regions of positively charged amino acids while the nuclear export signals tend to be short regions of hydrophobic amino acids. That is how the importins and exportins recognize the specific compartments on the molecules. Now inside the nucleus are chromosomes. We will talk about how chromosomes are packaged from DNA to a specific chromosome. If you take the 23 chromosomes that humans have and you take all the protein off of them and you line them up end to end, you get a piece of DNA that is approximately 2 meters long. Now that 2 meters of DNA has to fit into a nucleus of a diameter of about 10 µm. So an enormous amount of packaging has to go on to get the DNA into the nucleus and there also has to be room for the other things that go on in the nucleus. Chromosomes or DNA exists in two clearly distinct states in a cell. One of those states is referred to as the decondensed state which is where chromatin has a very loose conformation. An active cell has its chromosomes in the decondensed state for most of the cell cycle (during interface) because this allows easy access to the DNA for various molecules that are associated with its metabolism such as RNA polymerase, transcription factors, and the enzymes for DNA replication and repair (eg bowl of spaghetti - You don't know if the spaghetti you see is a single spaghetti or part of a different piece of spaghetti). The 2nd chromosomal state is known as the condensed state. This occurs in a very short period of the cell cycle is when mitosis or meiosis is going on. When the chromosomes are in a condensed form, they can be seen through a microscope with a proper chromosomal structure. During the decondensed state, the chromosomes cannot be discerned as specific fibers. The condensed state is useful for cell division and chromosome segregation. To condense we need to take the 2m long fibres and shorten them in a way that each are discrete entities and the chromatin fibers will not tangle around one another and rip apart (Breadsticks). There is various levels of packaging that exist in order to facilitate the packaging of DNA into interphase chromosomes and them it's condensation into mitotic chromosomes. The first level of the packaging is the interaction of DNA with histones. Table 12 – 1 These are Calf Thymus histones but are the same as any other histones. Do not memorize this table. There are five classes of histones. There are trends in this table. H2A, H2B, H3 and H4 tend to be smaller molecules, and are referred to as the core histones and they're very conservative. H4 is the most highly conserved histone. This is seen in an example where they took Mendel peas and some mammalian cells and isolated the H for stone from each one of those very distant organisms and found that both 102 amino acids long and there are only 2 changes between a Garden pea and human so this shows that it is very highly conserved during evolution for some functional consideration. Histone H1 is known as the linker histone and tends to be much less conserved. In organisms that are very closely related to one another, there is some similarity between the H1 but not nearly as much as in the core histones. The %Arg and the %Lys are the abbreviations for the amino acids arginine and lysine. These are the only two of the possibly three positively charged amino acids which is important in how packaging takes place. All of these histone molecules are rich in either arginine or lysine which helps them to do their job. Packaging of proteins such as histones with the DNA involves several levels of interaction. One of the most important of these is based on the bonds that occurred between negatively charged phosphates of DNA and the positively charged lysine and arginine of histones. The large amount of positive charge in histone molecules affectively conduces charge nuclearization so the negative phosphates of DNA don't repel one another. There are also hydrogen bonds that take place between the DNA backbone and the amino acids of
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